Tissue plasminogen activator (tPA) can be prepared by culturing cells which produce it, either on a large or small scale. The cells may produce tPA either natively, or by virtue of transformation with the recombinant gene. The colon fibroblast cell line, CCD18Co, isolated from the colon mucosa of a neonate, is available from the ATCC as accession no. CRL1459. This strain is known to produce tissue plasminogen activator as its normal cellular product. Various techniques are available for extending the life of these normally tPA-secreting cells. See, for example, Chang, S. E., Biochem Biophys Acts (1986) 823:161-194; Rhim, J. S., Science (1985) 227:1250-1252; Graham, F. L., J Gen Virol (1977) 36:59-72; Moyer, M. P., In Vitro Cellular and Developmental Biology (1987) 23:141-146.
In addition, mammalian cell lines, such as murine and Chinese hamster ovary cells, have been transfected with vectors bearing expression systems for tPA and obtained as stable transformed lines capable of producing this protein. See, for example, published EPO applications 117,059 and 117,060.
Methods are also available for culture of mammalian cells on a practical scale. Particularly advantageous is the use of a static maintenance reactor corresponding to that described in U.S. Pat. No. 4,537,860. Other techniques for culturing mammalian cells include use of beads for anchorage dependent cells, utilization of large-scale fermenters, roller bottles, and various perfusion reactors.
It is generally found that the levels of tPA produced by recombinantly modified mammalian cells are much higher than those obtained from cells which produce this protein normally. To some extent, the levels of tPA obtained are dependent on the nature of the culture medium, and it has been considered advantageous to supplement basal medium with serum. Even this supplementation, however, does not increase the level of production from non-recombinant cells to that obtainable using recombinant cells; also, it creates problems with respect to purifying the tPA product away from the serum proteins. The present invention suggests a culture medium which permits high levels of production of tPA both by recombinant and native producers.